Hepatocellular carcinoma (HCC) is a primary malignant cancer of the liver that most often develops in the background of liver cirrhosis (1,2). Hepatitis C virus (HCV) infection remains the major cause of liver cirrhosis and HCC among US Veterans (3,4). Recent advances in antiviral treatment have allowed an increasing number of cirrhotic patients to be cured with interferon (IFN)-free direct-acting antivirals (DAAs) (5,6). In the coming years, hepatitis C eradication is expected to decrease, but not eliminate, the risk of HCC among cirrhotic patients (7,8). The risk of HCC remains elevated post HCV clearance among patients with advanced fibrosis or cirrhosis and is also magnified by other risk factors such as daily alcohol intake, obesity, metabolic syndrome, diabetes, and race (African Americans or Hispanic) (7-16). HCC detected at an early stage can be effectively treated with transplantation, resection or even loco-regional therapies, but HCC detected at a late stage has a poor prognosis. The current HCC surveillance methods include liver ultrasound every six months with option of serum AFP (AASLD guideline). However, these methods lack the specificity and sensitivity needed to detect HCC at an early stage. Therefore, the development of a noninvasive, serum-based biomarker will allow early HCC detection and effective therapeutic intervention. This multicenter grant application proposes to test the performance of liver- specific serum biomarkers, together with standard-of-care tests including liver ultrasound, alpha-fetoprotein (AFP) AFP-L3 and DCP for HCC detection to monitor HCC risk among cirrhotic patients. This validation study will be performed by enrolling 2000 cirrhotic VA patients (HCV and non-HCV) from six different VA medical centers. Based on the incidence rate of HCC among the cirrhotic population, we anticipate >50 HCC cases will be detected at an early stage. Our hypothesis is that liver cirrhosis patients who do not resolve the ER-stress response will remain at an increased risk of HCC and therefore continue secreting HCC-specific exosomes that are enriched in glypican-3 and HSC70 (heat shock cognate protein 70) into the blood. Since the stress response depletes miRNA-122 transcription, we postulate that measurement of serum miRNA-122 levels will provide a powerful organ-specific biomarker for early prediction of cirrhosis regression after HCV cure. This application will explore the performance of HCC-specific exosome biomarkers, miRNA-122 and their additional benefit to serum AFP testing and liver ultrasound to improve surveillance of HCC and early detection of HCC. This hypothesis will be tested using the following Specific Aims: Aim 1 will determine the performance of serum exosomal cargoes (glypican-3, HSC70 and CD63) along with liver ultrasound, AFP, AFP-L3, DCP as a combined biomarker platform for the early detection of HCC among Veterans with liver cirrhosis after HCV cure using a longitudinal multicenter study. Aim 2 will develop a longitudinal multicenter study to determine the benefit of early HCC surveillance using the combination of exosomal biomarkers (glypican-3, HSC70 and CD63), serum miRNA-122 and non-exosomal (liver ultrasound, AFP, AFP-L3, DCP) for the early detection of HCC among Veterans with liver cirrhosis related to non-alcoholic fatty liver disease (NAFLD). Aim 3 will investigate whether measurement of liver specific miRNA- 122 levels in the serum along with noninvasive tests (FIB-4 index, APRI score and FibroScan) could increase the power of early assessment of cirrhosis regression or risk for persistent cirrhosis after HCV cure among Veterans with liver cirrhosis. If this application is selected for funding an exosome-based biomarker platform will be developed for early HCC detection among cirrhotic VA patients.